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. Author manuscript; available in PMC: 2019 May 15.
Published in final edited form as: Eur J Pharmacol. 2018 Mar 9;827:32–40. doi: 10.1016/j.ejphar.2018.03.013

In vitro and in vivo functional profile characterization of 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(isoquinoline-3-carboxamido)morphinan (NAQ) as a low efficacy mu opioid receptor modulator

Samuel Obeng a, Yunyun Yuan a, Abdulmajeed Jali b, Dana E Selley b, Yan Zhang a,*
PMCID: PMC5890425  NIHMSID: NIHMS954689  PMID: 29530590

Abstract

Evidence has shown that downstream signaling by mu opioid receptor (MOR) agonists that recruit β-arrestin2 may lead to the development of tolerance. Also, it has been suggested that opioid receptor desensitization and cyclic AMP overshoot contributes to the development of tolerance and occurrence of withdrawal, respectively. Therefore, studies were conducted with 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(isoquinoline-3-carboxamido)morphinan (NAQ), a MOR selective partial agonist discovered in our laboratory, to characterize its effect on β-arrestin2 recruitment and precipitation of a cyclic AMP overshoot. DAMGO, a MOR full agonist dose-dependently increased β-arrestin2 association with the MOR, whereas NAQ did not. Moreover, NAQ displayed significant, concentration-dependent antagonism of DAMGO-induced β-arrestin2 recruitment. After prolonged morphine treatment of mMOR-CHO cells, there was a significant overshoot of cAMP upon exposure to naloxone, but not NAQ. Moreover, prolonged incubation of mMOR-CHO cells with NAQ did not result in desensitization nor downregulation of the MOR. In functional studies comparing NAQ with nalbuphine in the cAMP inhibition, Ca2+ flux and [35S]GTPγS binding assays, NAQ did not show agonism in the Ca2+ flux assay but showed partial agonism in the cAMP and [35S]GTPγS assays. Also, NAQ significantly antagonized DAMGO-induced intracellular Ca2+ increase. In conclusion, NAQ is a low efficacy MOR modulator that lacks β-arrestin2 recruitment function and does not induce cellular hallmarks of MOR adaptation and fails to precipitate a cellular manifestation of withdrawal in cells pretreated with morphine. These characteristics are desirable if NAQ is pursued for opioid abuse treatment development.

Keywords: NAQ, mu opioid receptor, opioid addiction, antagonist, β-arrestin2 recruitment, nalbuphine

1. Introduction

It is estimated that a total of 246 million people illegally use drugs worldwide, of this number, 32.4 million abuse opioids (United Nations Office on Drugs and Crime, 2015). In the United States, an estimated 4.4 million people aged 12 or older suffer from substance use disorders related to prescription opioids and an estimated 435,000 are addicted to heroin (Center for Behavioral Health Statistics and Quality, 2015). Opioid addiction has devastating effects on societies and an alarming observation is that opioid misuse has been on the rise recently; the number of unintentional overdose deaths from opioid prescription analgesics has soared in the United States, more than quadrupling since 1999 (Muhuri et al., 2013).

Currently, drugs used to treat opioid addiction include opioid agonists methadone and buprenorphine. However, 40–60 % of patients on these drugs relapse (National Institute on Drug Abuse, 2012). Interestingly, opioid receptor antagonists such as naltrexone and naloxone have been shown to block relapse and curb drug craving in opiate addicts (Chen et al., 2010; George and Ekhtiari, 2010; Minozzi et al., 2011). On the other hand, some severe side effects have been reported with these drugs. For example, patients receiving naltrexone for opioid dependence reported depression, dysphoria and showed high rates of overdose and suicide (Miotto et al., 2002; Ritter, 2002). In addition, naloxone at high doses has been found to cause pulmonary edema and cardiac arrhythmias (van Dorp et al., 2007). Studies have indicated that the observed side effects may be due to the lack of selectivity for the mu opioid receptor (MOR) over other opioid receptors, particularly the delta and kappa opioid receptors (DOR and KOR) (Miotto et al., 2002; Ritter, 2002; van Dorp et al., 2007). Moreover, studies using MOR knockout mice have shown that the dependence and abuse liability, respiratory depression, and constipation associated with opioids were abolished, indicating that the addiction and abuse liability of opioids are mainly mediated through the MOR (Gavériaux-Ruff and Kieffer, 2002; Matthes et al., 1996; Skoubis et al., 2001). Therefore, a highly selective MOR antagonist would be an advantageous agent to treat opioid addiction with fewer side effects than naltrexone and naloxone.

A highly selective MOR ligand 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(isoquinoline-3-carboxamido)morphinan (NAQ), was recently identified in our laboratory (Fig. 1). NAQ showed an affinity of 0.55 nM to MOR with over 200-fold selectivity for the MOR over the DOR and 50-fold selectivity over the KOR (Yuan et al., 2011). NAQ acted as a low-efficacy MOR partial agonist in the [35S]GTPγS binding assay, but antagonized the effects of DAMGO (a MOR full agonist) and morphine in the [35S]GTPγS binding assay and warm-water tail immersion assay (Cornelissen et al., 2018; Li et al., 2009; Siemian et al., 2016; Yuan et al., 2011, 2013, 2015). Further pharmacological characterization showed that NAQ significantly reversed morphine withdrawal-associated depression of intracranial self-stimulation (ICSS) in rats (Altarifi et al., 2015). We herein report further characterization of NAQ to obtain a more comprehensive pharmacological profile, which indicated that NAQ does not produce significant cellular responses associated with opioid withdrawal, and make it a promising candidate for further development to treat opioid abuse and addiction.

Fig. 1.

Fig. 1

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NAQ together with drugs used to treat opioid addiction.

2. Materials and Methods

2.1 [35S]GTPγS binding assay

Membranes were prepared from human MOR-CHO (hMOR-CHO) cells and mouse MOR-CHO cells (mMOR-CHO). Ligand-stimulated [35S]GTPγS binding was performed as described previously (Selley et al., 1998, 1997). Briefly, membranes (10 μg of protein) were incubated with 0.1 nM [35S]GTPγS (specific radioactivity was 1250 Ci/mmol) and 10 μM GDP for 90 min at 30 °C with or without varying concentrations of indicated ligand in assay buffer (50 mM Tris-HCl, 3 mM MgCl2, 100 mM NaCl, 0.2 mM EGTA, pH 7.4). Nonspecific binding was determined with 20 μM unlabeled GTPγS and basal binding was determined in the absence of MOR ligand. A sample containing 3 μM DAMGO was included in each assay to determine maximal stimulation by a full agonist at the MOR. The incubation was terminated by rapid filtration through GF/B glass fiber filters and rinsed three times with ice-cold wash buffer (50 mM Tris-HCl, pH 7.2). Bound radioactivity was determined by liquid scintillation spectrophotometry at 95% efficiency for 35S. Net-stimulated [35S]GTPγS binding was defined as ligand-stimulated minus basal binding. Percent stimulation was defined as (net-stimulated/basal [35S]GTPγS binding) × 100%. Percent DAMGO-stimulated [35S]GTPγS binding was defined as (net-stimulated binding by ligand/net-stimulated binding by 3 μM DAMGO) × 100%.

2.2 Calcium flux assay

hMOR-CHO cells were maintained as described previously (Zhang and Xie, 2012). Four h after Gαqi5 transfection, cells were plated at 30,000 cells per well into a clear bottom black walled 96-well plate (Greiner Bio-one) and incubated for 24 h. The growth media was then decanted, and the wells were washed with 50:1 HBSS: HEPES assay buffer. Cells were then incubated with either 80 μl (agonism study) or 55 μl (antagonism study) of Fluo4 loading buffer (40 μl, 2 μM Fluo4-AM (Invitrogen), 84 μl of 2.5 mM probenecid, in 8 or 5.5 ml of assay buffer) for 30 min. For antagonism studies, 25 μl of varying concentrations of test compounds were added in triplicate and the plate was incubated for an additional 15 min. Plates were then read on a FlexStation3 microplate reader (Molecular Devices) at 494/516 ex/em for a total of 120 s. After 15 s of reading, 20 μl of varying concentrations of test compounds in triplicate (agonism study) or 500 nM of DAMGO (NIDA, antagonism study) in assay buffer, or assay buffer alone (control), were added. Changes in Ca2+ flux were monitored and peak height values were recorded. The obtained values were then subjected to nonlinear regression analysis to determine EC50 or IC50 values using GraphPad Prism 6.0 (GraphPad Software, San Diego, CA).

2.3 cAMP accumulation assays

2.3.1 Inhibition of adenylyl cyclase (AC)

Cells were plated at a density of 3 × 105 cells per well in 24-well plates and allowed to double overnight. For assay, the media was replaced by serum-free DMEM/F12 containing 20 mM HEPES, pH 7.3, the phosphodiesterase inhibitors, RO-20-1724 (0.1 mM) and isobutylmethylxanthine (0.1 mM), and 2% BSA (reaction buffer), and cells were incubated for 30 min at 37 °C. Forskolin was then added to a final concentration of 10 μM to all samples (except the basal and blank conditions) with or without varying concentrations of the indicated opioid ligand or an equivalent volume of reaction buffer to a final volume of 0.2 ml, and the reaction was initiated by transferring the plate to 37 °C water bath. The incubation was conducted for 8 min at 37 °C, and the reaction terminated by transferring the plate to an ice bath. Cells were immediately lysed by replacing the incubation solution with 3% perchloric acid. Samples were incubated on ice for 30 min, neutralized with 15% potassium bicarbonate and subjected to centrifugation at 1,000 × g for 10 min to isolate the precipitate.

cAMP levels were determined in aliquots of the supernatant by competitive binding of unlabeled cAMP from samples with [3H]cAMP (specific radioactivity was 26.4 Ci/mmol) to a cAMP binding protein (PKA regulatory subunit). Briefly, cell supernatant or cAMP standards were combined with [3H]cAMP and cAMP binding protein in a TRIS-EDTA buffer and incubated on ice for 90 min. A blank (no cells), cell blank (with cells), and total binding were assayed in the absence of cAMP binding protein. Unbound cAMP was removed through the addition and subsequent centrifugation (15,000 × g, 10 min, 4 °C) of 100 ml of a charcoal/dextran suspension. Radioactivity of the supernatant was determined using liquid scintillation spectrophotometry at 45% efficiency for [3H]. A log transformation calibration curve of radioactivity versus standards was generated on Microsoft Excel.

2.3.2 AC sensitization assay

mMOR-CHO cells were incubated in serum-free DMEM/F12 for 4 h with 5 μM morphine, and the pretreatment was terminated by removal of the morphine-containing media, and washing twice with serum-free DMEM/F12. Cells were then incubated in reaction buffer for 25 min at 37 °C followed by the addition of indicated ligand to a final concentration of 1 μM, and the incubation continued for another 5 min. Forskolin was then added to a final concentration of 10 μM and final volume of 0.2 ml, and samples were incubated for 8 min at 37 °C. The reaction was terminated and cAMP accumulation was quantified as described in paragraph two of section 2.3.1. Overshoot was calculated as the percentage of forskolin-stimulated cAMP accumulation in the absence of the indicated drug, i.e. vehicle.

2.4. Chronic treatment of mMOR-CHO cells with opioid ligands

2.4.1 Treatment of mMOR-CHO cells with opioid ligands

mMOR-CHO cells were grown in culture media (DMEM/F12 media, 10% FBS, 1% penicillin/streptomycin, 0.5% G418) for 5 days in an incubator set at 30 °C with 5% CO2 and 95% humidity. On the fifth day when the cells were confluent, the culture media was removed and the cells were rinsed with 5 ml PBS. The cells were then treated with DAMGO (5 μM), morphine (5 μM), nalbuphine (1 μM), NAQ (1 μM), naltrexone (1 μM) and vehicle (0.02% DMSO) dissolved in DMEM/F12 media and incubated for 24 h. After incubation, the treatment media was removed and the cells were washed three times with 10 ml phosphate-buffered saline (PBS). 5 ml PBS was added to each dish and the cells were then scraped off the dishes using a scraper. The cells were then centrifuged at 1,000 × g for 10 min. After centrifugation, the supernatant was decanted and membrane buffer (50 mM Tris, 3 mM MgCl2, and 1 mM EGTA, pH 7.4) was added to each sample. The cells were then homogenized and then centrifuged again at 50,000 × g for 10 min. The supernatant was decanted and the cells homogenized again in membrane buffer. A Bradford assay was conducted to determine the concentration of the membrane protein. The membrane protein preparations were then stored at −80 °C.

2.4.2 mMOR receptor saturation assay

Membranes were homogenized in membrane buffer and centrifuged at 50,000 × g for 10 min. This step was repeated to ensure that the drugs were completely removed from the receptor. The supernatant was then decanted and membranes were re-suspended in 50 mM Tris, 3 mM MgCl2, and 0.2 mM EGTA (pH 7.4). A Bradford assay was conducted to determine the protein concentration. The MOR membrane protein (30 μg) was then incubated with varying concentrations of [3H]naloxone (specific activity = 66.58 Ci/mmol) for 90 min at 30 °C. Nonspecific binding was determined using 5 μM naltrexone. The incubation was terminated by rapid filtration and bound radioactivity was determined as described in section 2.1. KD and Bmax values were determined by non-linear regression using GraphPad Prism 6.0 (GraphPad Software, San Diego, CA).

2.5 Beta-arrestin2 recruitment assay

This experiment was performed as described in the manufacturer’s protocol. Briefly, human CHO-K1 cells (DiscoveRx) were plated into a 96-well tissue culture plate and then incubated for 24 h at 37 °C, 5% CO2. For agonism studies, 10 μl of test compounds were added in duplicate and incubated for 90 min at 37 °C. For antagonism studies, 5 μl of test compounds were first added in duplicate and incubated for 30 min at 37 °C. Then 5 μl of DAMGO at 1 μM (~22 fold EC80 of DAMGO in the agonism study) was added and the cells were incubated for 90 min at 37 °C. After the 90 min incubation time, 55 μl of detection reagent was added to each well and incubated for 60 min at ambient temperature (25 °C). The luminescence was then read on a FlexStation3 microplate reader (Molecular Devices) for 200 ms. The obtained values were then subjected to nonlinear regression analysis to determine EC50, Emax or IC50 values of test compounds using GraphPad Prism 6.0 (GraphPad Software, San Diego, CA).

2.6 In vivo studies

All procedures were carried out in accordance with the “Guide for the Care and Use of Laboratory Animals” (Institute of Laboratory Animal Resources, National Academy Press, 1996) and were approved by the Institutional Animal Care and Use Committee of Virginia Commonwealth University. The animal facilities have been certified by the American Association for the Accreditation of Laboratory Animal Care. ICR male mice (Harlan, Inc., Indianapolis, IN) weighing 20 to 30 g were housed in groups of five with free access to food and water. Each animal was tested only once and all solutions were prepared in sterile water for injection. Six mice per treatment regimen were used. The ED50 or ED80 and AD50 values and their respective confidence limits were calculated by least squares linear regression analysis using the method and program adapted from Tallarida and Murray (1987).

2.6.1 Tail-flick assay

Antinociception was assessed by the tail-flick method described by D’Amour and Smith, as modified by Dewey and co investigators (D’Amour and Smith, 1941; Dewey et al., 1970). Briefly, the mouse’s tail was placed in a groove, which contained a slit under which was located a photoelectric cell. When the heat source of noxious stimulus was turned on, the heat focused in the tail, and the animal responded by flicking its tail out of the groove. Light thus passed through the slit and activated the photocell which, in turn stopped the recording timer. A control response (2–4 s) was determined for each mouse. To minimize tissue damage a maximum latency of 10 s was imposed. Antinociceptive response was calculated as percent maximal possible effect (%MPE), where %MPE = [(test – control)/(10 – control)] × 100. The mice were tested 20 min after the s.c. administration of agonist. In the TF versus morphine study, percent antagonism was calculated as [1 – (antagonist + agonist MPE)/(agonist MPE)] × 100. NAQ was given 10 min before receiving the morphine ED80 dose. Testing occurred 20 min later.

2.6.2 Phenylquinone (PPQ) abdominal-stretching assay

This experiment was conducted as reported previously (Pearl and Harris, 1966). The mice were injected with test drug and 10 min later received 2.0 mg/kg intraperitoneally (i.p.) of a freshly prepared PPQ solution. The mice were then placed in cages in groups of three each and 10 and 15 min after the PPQ injection, the total number of stretches per group were counted over 1 min period. A stretch was characterized by an elongation of the mouse’s body, development of tension in the abdominal muscles, and extension of the hindlimbs. The antinociceptive response was expressed as the percent inhibition of the PPQ-induced stretching response.

2.7 Data Analysis for receptor signaling assays

All experiments were performed in duplicate and data were reported as mean values ± S.E.M. from at least three independent determinations. Ligand concentration-effect curves were fit by iterative non-linear regression analysis using GraphPad Prism 6.0 (GraphPad Software, San Diego, CA). Statistical significance was determined by the two-tailed Student’s t-test or ANOVA with the indicated post-hoc test, performed using GraphPad Prism 6.0.

3. Results

3.1 [35S]GTPγS binding

To determine the potency and relative efficacy of NAQ to activate G-proteins, concentration-effect curves of ligand-stimulated [35S]GTPγS binding were examined. In this experiment, a CHO cell line engineered to express relatively higher levels of the hMOR than previously reported (Yuan et al., 2011) were used to determine the relative efficacy and potency of NAQ (Fig. 2A). This cell line expressed approximately the same MOR Bmax value (~2–4 pmol/mg) as the mMOR-CHO cells in which most of the in vitro pharmacological characterization of our compounds was conducted previously (Li et al., 2009; Yuan et al., 2013). The potency and efficacy of NAQ was also examined using these mMOR-CHO cells here (Fig. 2B). In both cell lines, NAQ was compared with the MOR agonist morphine, antagonist naltrexone, and a low efficacy partial agonist nalbuphine. The reason for comparing to nalbuphine was based on the fact that NAQ and nalbuphine were previously found in separate studies to exhibit relatively similar efficacies for MOR-mediated G-protein activation (Emmerson et al., 1996; Li et al., 2009; Selley et al., 1998). The stimulation for each compound was determined as the percent stimulation produced by the compound relative to the MOR full agonist DAMGO (3 μM). From the results, it was observed that morphine showed the highest relative Emax in both hMOR-CHO (83.9 ± 2.7 % DAMGO stimulation) and mMOR-CHO cells (95.0 ± 1.4 % DAMGO stimulation), indicating that morphine acted as an MOR agonist. On the other hand, naltrexone only showed 7.1 ± 0.9 % of DAMGO stimulation in hMOR-CHO cells and 5.9 ± 0.7 % of DAMGO stimulation in mMOR-CHO cells, indicating very low efficacy, consistent with its use as an MOR antagonist. NAQ and nalbuphine showed 14.4 ± 2.1 and 21.2 ± 0.9 % of DAMGO stimulation respectively in the hMOR-CHO cells and 21.4 ± 1.1 and 26.4 ± 1.6 % of DAMGO stimulation respectively in the mMOR-CHO cells (Fig. 2). It’s worth noting that the potency of morphine in the hMOR-CHO (EC50 = 110.5 ± 8.4 nM) cells was approximately 2-fold lower than that in the mMOR-CHO cells (EC50 = 51.4 ± 4.7 nM). The potencies of NAQ and nalbuphine were found to be 3.6 ± 0.6 nM and 30.2 ± 2.1 nM respectively in hMOR-CHO cells, while in mMOR-CHO cells they were found to be 6.2 ± 0.7 nM and 15.2 ± 1.8 nM, respectively. These results demonstrated that although NAQ was somewhat more potent than nalbuphine, both ligands stimulated MOR-mediated G-protein activation with similar intrinsic efficacy, and acted as low efficacy partial agonists relative to DAMGO. Furthermore, although modest differences in opioid ligand potency and relative efficacy values were obtained between the CHO cells lines expressing human or mouse MOR, the values were comparable between species of MOR.

Fig. 2.

Fig. 2

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[35S]GTPγS binding curve comparing morphine, nalbuphine, NAQ, and naltrexone in (A) human MOR-CHO cells and (B) mouse MOR-CHO cells. The data were presented as the mean ± S.E.M. (n = 4), normalized to the maximum stimulation caused by 3 μM DAMGO (100%; vehicle treatment = 0%).

3.2 Calcium flux

While [35S]GTPγS binding is a direct measurement of GPCR-mediated G-protein activation, the first step in MOR signaling, cytosolic Ca2+ concentration can be measured as a downstream secondary messenger. This may allow for amplification of the signal leading to a higher level of partial agonism compared to [35S]GTPγS binding (Zhang and Xie, 2012). In other words, measurement of cytosolic calcium concentration is an indirect way to determine GPCR function. The results revealed that whilst DAMGO concentration-dependently increased intracellular Ca2+ levels, NAQ and nalbuphine did not show any apparent agonism even at the highest concentration tested of 100 μM (Fig. 3A). When the assay period was extended to 10 min, NAQ and nalbuphine still did not show any apparent agonism. (Fig. 3B). On the other hand, both NAQ and nalbuphine concentration-dependently inhibited Ca2+ flux induced by DAMGO (Fig. 3C), similar to the MOR antagonist naltrexone, but with lower potencies.

Fig. 3.

Fig. 3

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Ca2+ flux assays in Gαqi5 transfected hMOR-CHO cells. (A) The MOR full agonist DAMGO concentration-dependently increased intracellular Ca2+ level, whereas no apparent agonism was observed for NAQ and nalbuphine. (B) The detection time extended to 10 min in contrast to 2 min. (C) NAQ and nalbuphine antagonized DAMGO-induced intracellular Ca2+ increase. The Ki ± S.E.M. values of NTX, NAQ and nalbuphine are: 0.21 ± 0.06 nM, 3.51 ± 0.35 nM, and 14.72 ± 2.06 nM, respectively. Data are presented as mean values ± S.E.M. (n = 3).

3.3 Chronic treatment of mMOR-CHO cells with opioid ligands

Continuous exposure of opioid receptors to agonists may lead to cellular tolerance caused by desensitization of the receptor. Desensitization is due to uncoupling of the receptor from G-protein activation, and can be followed by downregulation, measured as a decrease in the receptor Bmax value, whereas antagonists or inverse agonists can produce sensitization and/or upregulation. Therefore, a study was conducted to determine whether NAQ, nalbuphine, naltrexone, morphine, and DAMGO produced any of these adaptations of the MOR. The KD and Bmax values of [3H]naloxone saturation binding was determined in membranes from mMOR-CHO cells pretreated with these compounds. The results showed that the MOR agonists, DAMGO and morphine produced downregulation of the MOR, as indicated by reductions in the [3H]naloxone Bmax value relative to vehicle-treated cells, whereas NAQ, nalbuphine and naltrexone did not produce significant downregulation (Fig. 4). However, NAQ and naltrexone produced an approximate doubling of the [3H]naloxone KD value. Likewise, pretreatment with DAMGO or morphine, but not NAQ, nalbuphine or naltrexone, resulted in apparent desensitization of MOR-mediated G-protein activation in response to the full MOR agonist DAMGO. These results showed that the EC50 of DAMGO in membranes from DAMGO and morphine pretreated cells increased by approximately 4- and 5-fold, respectively, compared to vehicle treated cells, whereas DAMGO EC50 values were unaffected by pretreatment of cells with NAQ, nalbuphine or naltrexone (Fig. 5D). The DAMGO Emax value in DAMGO pretreated cells was 82.2% and in morphine pretreated cells was 107.5% compared to vehicle control cells, but they were not significantly different. The DAMGO Emax values in nalbuphine, NAQ and naltrexone pretreated cells were also not significantly different, from the value obtained in vehicle control cells (Fig. 5C). The receptor efficiency values were then calculated as the Emax/EC50 ratio of DAMGO in membranes from the pretreated cells. The results showed that DAMGO and morphine pretreatment significantly reduced receptor efficiency to a similar extent, whereas NAQ, nalbuphine and naltrexone did not (Fig. 5E). These results demonstrate that, similarly to other low efficacy ligands, prolonged exposure of cells to NAQ did not produce MOR adaptations that occur with high efficacy MOR ligands.

Fig. 4.

Fig. 4

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Saturation assay results for treated mMOR-CHO cells using [3H]naloxone showing (A) Bmax (pmol/mg) and (B) KD (nM) for each treatment. Data are presented as mean values ± S.E.M. (n = 4). * Indicates P < 0.05 compared to vehicle, while ** indicates P < 0.01 compared to vehicle.

Fig. 5.

Fig. 5

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Effects of ligand pretreatment on mMOR-stimulated [35S]GTPγS binding in mMOR-CHO cells. DAMGO stimulation in each cell preparation was normalized to the maximal stimulation produced by DAMGO in cells treated with vehicle, which was set to 100%. (A) DAMGO concentration-effect curves in vehicle, DAMGO and morphine treated cells. (B) DAMGO concentration-effect curves in vehicle, nalbuphine, NAQ and naltrexone treated cells. (C) DAMGO Emax values derived from the concentration-effect curves shown in panels A and B. (D) DAMGO EC50 values derived from the concentration-effect curves shown in panels A and B. (E) Ratio of DAMGO Emax/EC50 derived from the values shown in panels C and D. Data are presented as mean values ± S.E.M. (n = 4). * Indicates P < 0.05 compared to vehicle, while ** indicates P < 0.01 compared to vehicle.

3.4 β-Arrestin2 recruitment

Studies were conducted using the PathHunter system in human CHO-K1 cells to quantitatively determine whether NAQ would be functionally selective or show biased partial agonism towards G-proteins over β-arrestin2. The MOR full agonist DAMGO concentration-dependently increased β-arrestin2 association, whereas occupancy of the MOR by NAQ or nalbuphine did not recruit β-arrestin2, even at the highest concentration (10 μM) of ligand that was used (Fig. 6A). In fact, both NAQ and nalbuphine antagonized DAMGO-induced β-arrestin2 recruitment in a concentration-dependent fashion, with IC50 values of 10.4 ± 5.7 nM and 29.7 ± 5.0 nM, respectively (Fig. 6B).

Fig. 6.

Fig. 6

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β-Arrestin2 recruitment assay using PathHunter eXpress OPRM1 CHO-K1 β-arrestin2 assay kit. (A) The MOR full agonist DAMGO concentration-dependently increased β-arrestin2 association, whereas NAQ and nalbuphine displayed no apparent agonism. (B) NAQ and nalbuphine antagonized DAMGO-induced β-arrestin2 recruitment in a concentration-dependent fashion. Data are presented as mean values ± S.E.M. (n = 3).

3.5 AC inhibition and sensitization assay

It has been reported that long-term administration of opioid agonists causes a homeostatic sensitization of AC, which consequently produces a cAMP overshoot upon withdrawal of the opioid agonists (Charles and Hales, 2004; Koob and Bloom, 1988; Nestler and Aghajanian, 1997; Watts, 2002). Since the MOR is Gi/o coupled, activation of the receptor can result in the reduction of cAMP production, therefore we examined inhibition of AC activity by NAQ compared to DAMGO and nalbuphine in intact mMOR-CHO cells. Results showed that NAQ and nalbuphine inhibited AC less efficaciously than the MOR full agonist DAMGO, with nalbuphine being more potent than NAQ (Fig. 7A). Another study was conducted to determine whether NAQ precipitates withdrawal in morphine treated mMOR-CHO cells. In this experiment, it was observed that there was a significant overshoot of cAMP upon precipitation of withdrawal with naloxone, but not nalbuphine nor NAQ, compared to the vehicle (Fig. 7B).

Fig. 7.

Fig. 7

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cAMP accumulation assays. (A) Inhibition of 10 μM forskolin-stimulated cAMP by varying concentrations of the indicated ligand. Data are presented as mean % inhibition values ± S.E.M. (n = 3) of the percentage of cAMP inhibition, where stimulation by 10 μM forskolin alone is designated as 0%; (B) Morphine-mediated cAMP overshoot was precipitated by naloxone, but not NAQ and nalbuphine treatment. Data are presented as mean values ± S.E.M. (n = 3) of the percentage of forskolin-stimulated cAMP in vehicle-pretreated cells, which was set to 100%. * Indicates P < 0.05 compared to vehicle.

3.6 In vivo characterization of NAQ

NAQ was then further studied in mouse antinociceptive assays to characterize its in vivo agonist and antagonist properties. As indicated in Table 1, NAQ showed dose-dependent, but weak antinociceptive effects in the tail flick test. The maximum antinociceptive effect observed for NAQ at a dose of 30 mg/kg was 12% of the maximum possible effect (MPE) of 10 mg/kg morphine. In the same test, NAQ was active as a potent opioid antagonist with an AD50 value of 5.7 mg/kg. On the other hand, NAQ showed agonist activity in the PPQ test, with an ED50 value of 1.52 mg/kg. To determine whether kappa and delta opioid agonist activities contributed to the agonist effect of NAQ in the PPQ test, opioid receptor type-selective antagonists were administered prior to administration of an ED80 dose of NAQ. Table 2 shows that both the kappa antagonist norBNI and the delta antagonist naltrindole partially inhibited NAQ-induced antinociception over a portion of the dose range tested, such that 1 mg/kg naltrindole and 10 mg/kg norBNI produced the greatest inhibition (~65%) of NAQ’s agonist effects.

Table 1.

Effects of NAQ in the tail-flick and paraphenylquinone (PPQ) antinociceptive tests.

Antinociception Test ED50 or AD50 (95% C.L.) or % effect
1. Tail flick (agonism) 2% at 1 mg/kg, 9% at 10 mg/kg, and 12% at 30 mg/kg
2. Tail flick versus ED80 dose of morphine (antagonism) AD50 = 5.7 (1.92–17.37) mg/kg
3. PPQ (agonism) ED50 = 1.52 (0.77–3.0) mg/kg
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Table 2.

norBNI and naltrindole versus the ED80 dose of NAQ in the PPQ antinociception test.

NorBNI versus ED80 of NAQ Naltrindole versus ED80 of NAQ
norBNI % antagonism of NAQ Naltrindole % antagonism of NAQ
mg/kg, s.c. ED80 mg/kg, s.c. mg/kg. s.c. ED80 mg/kg, s.c.
0.3 0 0.3 22
1.0 33 1.0 67
3.0 22 3.0 22
10.0 63 10.0 0
30.0 44 30.0 0
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4. Discussion

4.1 [35S]GTPγS binding and Ca2+ mobilization

A CHO cell line engineered to express relatively higher levels of the hMOR than in our previous study (Yuan et al., 2011) was used to determine the relative efficacy and potency of NAQ compared to morphine, nalbuphine and naltrexone (Fig. 2A). The potency and relative efficacy of the compounds were also determined using mMOR-CHO cells (Fig. 2B). Not surprisingly, morphine (an agonist) produced the highest stimulation in both hMOR and mMOR-CHO cells whilst naltrexone (an antagonist) showed the lowest stimulation (Fig. 2A and B). NAQ and nalbuphine showed similar level of stimulation relative to DAMGO in both cell lines, indicating that NAQ and nalbuphine can act as partial MOR agonists. On the other hand, NAQ and nalbuphine did not show any significant agonism in the Ca2+ flux assay even when the assay time was increased to 10 min (Fig. 3). However, NAQ and nalbuphine inhibited Ca2+ flux induced by DAMGO (Fig. 3C) similar to the MOR antagonist naltrexone (NTX), indicating that NAQ can act as a MOR antagonist, depending on the signaling endpoint measured. One potential reason for NAQ not showing agonism may be that chimeric G proteins (Gαqi5) were used in the Ca2+ flux assay. A similar phenomenon was also observed with ipsapirone, a highly potent and efficacious 5-HT1A receptor agonist which did not show any agonism in the Ca2+ flux assay (Kostenis et al., 2005; Kowal et al., 2002). Another reason could be that NAQ and nalbuphine might have acted as slow binding ligands, and as a result, their activity may only be observable after 10 min, which is outside our experimental time course (Venkatakrishnan et al., 2013). Finally, being a low efficacy MOR ligand, Gi/o activation by NAQ may be too low to induce downstream Ca2+ release.

4.2 Chronic treatment of mMOR-CHO cells with opioids

Tolerance to opioids may result in part from downregulation or desensitization of the MOR (Allouche et al., 2014; Borgland, 2001; Dang and Christie, 2012; Sim-Selley et al., 2009; Williams et al., 2013). To determine whether NAQ produced MOR desensitization and downregulation in mMOR-CHO cells, a study was conducted whereby cells were incubated with NAQ for 24 h. This approach provided a suitable method to investigate mechanisms of cellular tolerance occurring at the receptor level. Opioid tolerance can be best quantified by the rightward shift in the dose-response curve that may be associated with a reduction in the maximum response. It was observed that incubation of the mMOR-CHO cells with MOR agonists, e.g. DAMGO or morphine, resulted in a rightward shift in the dose-response curve (Fig. 5A), and resulting in an increase in the DAMGO EC50 value (Fig. 5D). However, NAQ, nalbuphine, or naltrexone did not significantly change the potency nor efficacy of DAMGO compared to vehicle pretreatment (Fig. 5B, C and D). Thus, pretreatment with the opioid agonists morphine and DAMGO produced apparent desensitization of the MOR, whereas NAQ, nalbuphine and naltrexone did not. To determine whether downregulation of the MOR contributed to the apparent desensitization observed in the agonist-pretreated cells, [3H]naloxone saturation analysis was conducted (Christie, 2009; Koch and Höllt, 2008). DAMGO and morphine pretreatment caused a significant decrease in the MOR receptor density (Bmax) compared to vehicle-pretreated cells, while NAQ, nalbuphine, or naltrexone pretreatment did not significantly change the receptor density (Fig. 4A). Operational models used to quantify the loss of functional MOR-effector coupling in isolated systems after chronic morphine treatment have indicated a loss of approximately 80% of functional surface MOR would be required to account for the observed shift in agonist concentration-response curves (Bailey et al., 2009; Christie, 2009). Morphine and DAMGO pretreatment produced less than 80% reduction in MOR density, so it seems likely that the apparent desensitization produced by these agonists is not entirely due to receptor downregulation but due to loss of receptor efficiency (Fig. 5E). However, because we measured downregulation of total membrane-associated MOR, it is possible that a subset of these binding sites were internalized MOR in the vesicular fraction. Regardless of the exact mechanism, the key finding here is that pretreatment with NAQ and other low efficacy opioid ligands did not affect subsequent DAMGO-stimulated G-protein activation by the MOR. Interestingly, naltrexone and NAQ-pretreated cells showed a significant reduction in [3H]naloxone binding affinity (Fig. 4B). The reason for this effect is unclear, but could have been related to inadequate removal of these high affinity ligands from the MOR prior to the binding assay. However, pretreatment with these ligands did not result in any significant change in DAMGO potency in the functional assay, so it seems unlikely there was residual ligand present. Overall, the in vitro data obtained here suggest that NAQ may not produce MOR adaptations that contribute to tolerance.

4.3 β-Arrestin2 recruitment

Studies conducted with β-arrestin2 knockout (β-arrestin2-KO) mice showed that in mice lacking β-arrestin-2, desensitization of the MOR did not occur after chronic morphine treatment, and that these animals failed to develop antinociceptive tolerance (Bohn et al., 2000). In the current study, NAQ and nalbuphine were subjected to the β-arrestin2 recruitment assay using the PathHunter assay in CHO-K1 cells (DiscoveRx). It was observed that DAMGO, a MOR full agonist, dose-dependently increased β-arrestin2 association, whereas NAQ and nalbuphine displayed no apparent agonism (Fig. 6A). In contrast, NAQ and nalbuphine significantly antagonized DAMGO-induced β-arrestin2 recruitment in a dose-dependent fashion (Fig. 6B). These findings also suggest that NAQ may not produce tolerance. It has been shown that at high doses, chronic morphine treatment produces physical dependence in the β-arrestin2-KO mice similar to WT mice (Bohn et al., 2000; Raehal and Bohn, 2011). However at lower doses of morphine infusion, β-arrestin2-KO mice were protected from the onset of dependence as evidenced by a decrease in the severity of the antagonist-precipitated withdrawal response (Raehal and Bohn, 2011). This indicated that β-arrestin2 could be involved in the pathways leading to physical dependence. NAQ significantly antagonized DAMGO-induced β-arrestin2 recruitment (Fig. 6), which suggests that NAQ may inhibit the development of opioid dependence if co-administered with an opioid agonist. The results observed in this study further supported the data we observed in our previous study where NAQ did not induce β-arrestin2 recruitment and also NAQ pretreatment completely blocked DAMGO induced β-arrestin2 recruitment (Zhang et al., 2016, 2014). To be noted, in our previous study (Zhang et al., 2014), the β-arrestin2 recruitment assay was conducted using live cell confocal imaging, which was a qualitative assay. The β-arrestin2 recruitment assay using the PathHunter assay in CHO-K1 cells is a quantitative assay, and the results obtained using this platform corroborated the findings from our previous study.

4.4 AC inhibition and sensitization assay

NAQ behaved similarly to nalbuphine in the AC inhibition and AC sensitization assay (Fig. 7). The most encouraging observation was that NAQ did not precipitate cAMP overshoot in the AC sensitization experiment (Fig. 7B). It has been reported that long-term administration of opioid agonists causes a homeostatic sensitization of AC, which consequently produces a cAMP overshoot upon withdrawal of the opioid agonists. cAMP overshoot is proposed to model withdrawal as a consequence of opioid dependence, and the underlying sensitization of AC signaling may contribute to opioid dependence (Charles and Hales, 2004; Koob and Bloom, 1988; Nestler and Aghajanian, 1997; Watts, 2002). Furthermore, sensitization of the AC/cAMP pathway also represents a form of physiological tolerance (Avidor-Reiss et al., 1996; Christie, 2009). The results here showed that NAQ may have therapeutic potential in the prevention of opioid withdrawal.

4.5 In vivo characterization of NAQ

NAQ showed very weak antinociceptive activity in the tail flick assay but potent opioid antagonist effects in the same assay (Table 1). However, in the PPQ writhing assay, a model for visceral pain (Reichert et al., 2001), NAQ showed potent antinociceptive activity. To determine whether non-mu opioid receptors contributed to the agonist activity of NAQ, type-selective opioid receptor antagonists were administered. It was observed that naltrindole antagonized NAQ’s antinociceptive effect at a dose 10-fold lower than norBNI (Table 2). The biphasic effect of naltrindole on NAQ-induced antinociception was intriguing, as naltrindole has been shown to produce antinociception at very high doses (e.g. 10 mg/kg) (Jackson et al., 1989), which could account for the lack of antagonism at 10 and 30 mg/kg of naltrindole because naltrindole itself can produce antinociception at these doses. These results are consistent with our previous findings that, while the in vitro relative efficacy of NAQ at the KOR was low (13%), its efficacy at the DOR was moderate (54%) albeit with low potency (Yuan et al., 2011). All of this suggests that the NAQ doses tested may have achieved moderate KOR occupancy with low efficacy along with very low occupancy of DOR, but with higher efficacy. Thus, the antinociception to visceral pain observed with NAQ could have been due in part to activation of both the DOR and KOR.

5. Conclusion

NAQ, identified as a MOR-selective orthosteric ligand, was further characterized to obtain a more comprehensive pharmacological profile. NAQ produced very low maximal G-protein activation relative to DAMGO in both human and mouse MOR-expressing CHO cells. It was also observed that prolonged incubation of mMOR-CHO cells with NAQ did not produce desensitization or sensitization nor down- or upregulation of the MOR. NAQ also displayed no apparent agonism in the β-arrestin2 recruitment assay, but antagonized DAMGO-induced recruitment of β-arrestin2. Furthermore, NAQ did not precipitate cAMP overshoot in mMOR-CHO cells after prolonged exposure to morphine. In vivo studies conducted to further characterize receptor selectivity of NAQ revealed that it acted as an agonist in the PPQ test of visceral antinociception, likely due in part to partial agonism of the DOR and weak partial agonism of the KOR. In all, these results suggest that NAQ antagonizes or minimally activates MOR signaling, depending on the specific pathway tested, without producing MOR adaptation or precipitating cellular withdrawal, which makes it a promising candidate for future development as opioid abuse and addiction treatment.

Acknowledgments

This work was partially supported by NIH/NIDA DA024022 (Y.Z.) and NIDA Contract 7-8859. The authors thank Dr. Mario Aceto and Dr. Louis Harris for their generous help in the mouse PPQ study.

Footnotes

Declarations of interest

The authors declare no conflict of interest.

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